Mechanisms of Neural Crest Cell Development

描述(申请人提供)：心脏流出道的形成是一个复杂的形态发生过程，其失败会导致各种临床上重要的先天性心脏畸形。我们发现由PBX基因家族编码的同源结构域转录因子是小鼠心脏流出道发育所必需的。PBX基因突变导致永久性动脉干、法洛四联症、主动脉瓣和二尖瓣病变。这些缺陷类似于在鸡的神经脊细胞被切除时观察到的心脏异常。我们假设PBX基因对小鼠的神经脊细胞功能是必不可少的，并建议利用这些小鼠模型来研究Pbx1基因如何调节心脏流出道发育所需的神经脊细胞功能的机制。这些研究将为先天性心脏缺陷的发育机制提供深入的认识。具体地说，我们将研究(1)Pbx1是否在神经脊细胞内固有地作用于细胞以调节其功能，(2)Pbx1是否对决定神经脊细胞的发育命运至关重要，以及(3)Pbx1下游的分子通路在心脏发育中的作用。目的#1：确定心脏发育所需的Pbx1作用的细胞部位我们将利用基因Cre-lox方法和几个在神经脊细胞、心肌细胞和心内膜细胞中表达Ore重组酶的小鼠系来研究Pbx1可能调节心脏神经脊细胞发育的细胞部位。目的#2：确定Pbx1在神经脊细胞发育中的作用。为了研究是否需要Pbx1来确定心脏神经脊细胞的发育特性，我们计划使用RNA原位杂交和菱形分化标记来研究Pbx1/-小鼠胚胎中的菱形特性。由于心脏神经脊细胞起源于菱形核，因此菱形标记在PbxT1‘胚胎中的表达将为心脏神经脊细胞的命运决定提供新的线索。神经脊细胞的其他标记也将被用于研究神经脊细胞从神经管中迁移出来时的分化。目的#3：确定Pbx1缺失对分子通路的影响。我们将使用标准的分子生物学和胚胎学方法来研究Pax3通路在心脏发育中是否位于Pbx1的下游。

DESCRIPTION (provided by applicant): Formation of the cardiac outflow tract is a complex morphogenetic process, whose failure results in a variety of clinically important congenital heart malformations. We have found that the homeodomain transcription factors encoded by the Pbx gene family are required for development of the cardiac outflow tract in mice. Mutations in Pbx genes lead to persistent truncus arteriosus, tetralogy of Fallot, overriding aorta and bicuspid aortic valves. These defects resemble cardiac anomalies observed in chick when neural crest cells are ablated. We hypothesize that Pbx genes are essential for neural crest cell function in mice, and propose to use those mouse models to investigate the mechanisms of how Pbx1 gene regulates neural crest cell function required for cardiac outflow tract development. These studies will provide insights to the developmental mechanisms of congenital heart defects. Specifically, we will study (1) whether Pbx1 acts cell intrinsically within the neural crest cells to regulate their function, (2) whether Pbx1 is essential to program the developmental fates of neural crest cells and (3) the molecular pathways downstream of Pbx1 in cardiac development. Aim #1: Determine the cellular sites of Pbx1 action required for cardiac development We will use the genetic Cre-lox methods and several mouse lines that express Ore recombinase in neural crest cells, myocardial cells and endocardial cells to study the cellular sites where Pbx1 may function to regulate the development of cardiac neural crest cells. Aim #2: Determine the role of Pbx1 in neural crest cell development. To study if Pbx1 is required for specifying the developmental identities of cardiac neural crest cells, we plan to employ RNA in situ hybridization and rhombomeric differentiation markers to study the rhombomeric identities in Pbx1-/- mouse embryos. As cardiac neural crest cells originate from rhombomeres, expression of rhombomeric markers in the PbxT1' embryos will provide insights to the fate specification of cardiac neural crest cells. Additional markers of neural crest cells will also be used to study the differentiation of neural crest cells as they migrate out of the neural tube. Aim #3: Define the molecular pathways affected by the absence of Pbx1. We will use standard molecular biology and embryology methods to study if Pax3 pathway is downstream of Pbx1 in cardiac development.